Yeast chorismate mutase and other allosteric enzymes

Pure & Appl, Chem., Vol. 70, No. 3, pp. 527-531, 1998. Printed in Great Britain. (B 1998 IUPAC Yeast chorismate mutase and other allosteric enzymes Function William N. Lipscomb" and Norbert StratePb 'Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, MA 02138, USA bpresent address: Institut fur Mikrobiologie and Genetik Freie Universitdt Berlin, Takustrasse 6, 014195. Berlin, Germuny Abstract: Analysis of five crystal structures of dimeric yeast chorismate mutase include a T-state bound to the allosteric inhibitor Tyr, two R-state mutants and two super-r wildtype structures bound to the transition state inhibitor plus either Tyr or the allosteric activator Trp. Relative rotations of one subunit relative to the other are 0' (reference) for T, 15' for R and 22' for super-r states. A well defined pathway for conformational changes occurs from the active site to the subunit interface. Comparisons are made with other allosteric enzymes. YEAST CHORISMATE MUTASE (YCM) This enzyme, which converts chorismate to prephrenate in the pathway to Tyr and Phe, is inhibited at an allosteric site by Tyr and activated by Trp (ref. 1) (Fig. 1). Chorismate is also a substrate for anthranilate synthase which leads to Trp, and which is allosterically inhibited by Trp. Such regulatory processes achieve a balance between aromatic amino acids based on pyrmidine or purine rings. These enzymes are present only in archaebacteria, eubacteria, plants and fungi, thus providing an opportunity to design antibacterials, herbicides or fungicides which may be tolerated by humans. 'OOC, -0oc f y o - hoacoo- - I OH,+- - O o C p ( coo- - HO OH Chorismate Transition State Prephenate HOi Fig. 1. The reaction catalyzed by YCM. A transition state analogue is also shown. Structure The T-state dimer of YCM (ref. 2), 2 X 30k Da, consists mostly of helices and connecting loops (Fig. 2). The dimer axis lies in the plane of the paper, and the allosteric axis is perpendicular to the plane of the paper and 527

528 w. N. LIPSCOMB AND N. STRATER intersects the dimer axis. The allosteric axis rotations described in the Abstract move the two helices Hz closer together at the subunit interface and make them more nearly parallel. Four helices Hz, Hs, HI1 and Hlz (Fig.2) surround the active site and contribute binding and catalytic site chains. As the transition state is approached, the negative charge developing on the ether oxygen 02' (Fig. 1) is stabilized by Lys 168 and by a protonated Glu 246, thus stretching the CYO'Z bond and initiating, the Claisen rearrangement of the enolpyruvate unit to yield prephrenate (Fig. 1). The Transition Fig. 2. The structure of dimeric YCM, showing two activator Trp molecules and two transition state inhibitor molecules I. In the T to super-r transition, which is dominated by the substrate (here the transition state analogue), a series of changes propagate from the substrate site to the subunit interface (Fig. 3). As Arg 157 binds to the substrate the Arg 157... Glu 23 interaction is broken, and Glu 23 then moves to interact with Arg 204 and Lys 208 ofh1l. Also Lys 208 bonds to Asp 24 which binds to Tyr 212 near the interface, influencing Phe 28 (Fig. 3). These changes induce the relative rotation of subunits, and thus activate the active site of the other subunit. The transition from the relatively tight T state to the looser R state is also promoted by the large aromatic ring system of the activator Trp, and inhibited by the smaller ring of the allosteric inhibitor Tyr. In addition the Trp inhibition involves the region of Hi plus the 80's loop (Fig. 2 and 4). In this region there is one hydrogen bond to the tyrosine OH group. This OH group of Tyr also donates a hydrogen bond to the hydroxyl group of Thr 145 of the & helix. The allosteric activator Trp cannot make these two hydrogen bonds, which favor the T state when Tyr is present at the allosteric site. Thus, Tyr favors the T state, whereas Trp favors the R state. These allosteric effectors have less influence than does the transition-state inhibitor (and therefore, presumably, the substrate). Rotations of subunits COMPARISON WITH OTHER ALLOSTERIC ENZYMES Similar rotations of subunits in allosteric transitions (ref 3) are seen in aspartate transcarbamylase (cgr6) where one c3 rotates 12" relative to the other c3, and each rz rotates by 15" (ref. 4). Also, in fructose-1,6- bisphosphatase (c4) one CLZ rotates by 17" (ref.5). In phosphohctokinase (~14) one CLZ rotates by 7" (refs. 6 0 1998 IUPAC, Pure and Applied Chemistry 70,527-531

Yeast chorismate mutase 529 and 7). In glycogen phosphorylase (a~) one monomer a rotates by 10" (ref. 8). In hemoglobin (a$*) one ap rotates by 15' (ref 9-11). In allosteric bacterial lactate dehydrogenase (a4) rotations occur of a2 about two axes by 6' and by 4" (ref. 12). Except for the increase of 1lA between c3's in aspartate transcarbamylase, relative translations are restricted to only a few A. The generality of these rotations leads to a suggestion that detailed studies of torques in allosteric transitions may be worth investigation. Fig. 3 Conformational changes of the T to R transition (arrows) between helices HZ and H11 extending from the active site to the dimer interface. Preservation of organized structure First, there is no significant change in the overall folding of the protein structures of allosteric proteins. Indeed, the transmission of conformational information requires organized secondary and tertiary protein 0 1998 IUPAC, Pure and Applied Chemistry70,527-531

530 w. N. LIPSCOMB AND N. STRATER structure in all allosteric enzymes studied so far. There are small changes, for examples the loss of one turn of helix (155-162) in phosphofructokinase and the coil to helix transition of the N-terminal sequence containing the phosphorylated Ser 14 in glycogen phosphorylase. Structure is well preserved in yeast chorismate mutase, and the long helix Hs connects the active site of one monomer to the allosteric site of the other monomer (ref 13). Although the symmetry before and after these allosteric transitions is largely preserved, partial occupancy of active or allosteric sites may lower symmetry during the transition. H4IL80s H HN H N A N w A 7 5 J H H H Ile A74 Fig. 4. The regulatory site in the region of the inhibitor Tyr (left, T state) and the activator Trp (right, R state). Letters A and B identify subunits. Chanpe of afinitv or change of Vniax Allosteric chorismate mutase shows mixed behavior in these changes during the allosteric transition. For example, the allosteric inhibitor 8 lowers both [S]O.~ and Vmax (ref. 14). In D-3-phosphoglycerate dehydrogenase Vmax is altered (ref.15). However, it is almost a generalization (ref3) that substrate affinity is increased in the T to R transition, as it is in aspartate transcarbamylase, phosphofructokinase, glycogen phosphorylase, allosteric lactate dehydrogenase, and hemoglobin. In fructose-l,6-bisphosphatase it is the affinity for one of the catalytic metal ions that is altered. Sharing of ligand sites favors concerted transitions This sharing occurs between adjacent subunits in aspartate transcarbamylase, fructose-l,6-bisphosphatase, phosphofructokinase and allosteric lactate dehydrogenase. In yeast chorismate mutase, the allosteric site is shared between the two subunits (Fig. 4). Inhibitors and activators share parts of the same regulatory site in aspartate transcarbamylase, bacterial phosphofructokinase, glycogen phosphorylase b (nonphosphorylated), and allosteric chorismate mutase. Dominance of substrate or allosteric effectors Substrates induce the T to R transition in hemoglobin, aspartate transcarbamylase and glycogen phosphorylase, and dominate this transition in yeast allosteric chorismate mutase. On the other hand, an allosteric effector causes the allosteric transition in fructose-l,6-bisphosphatase, phosphofructokinase and allosteric lactate dehydrogenase. Flexible R-state structures The angle increase from T (0') to 15" (R state) and 22" (super-r state) for yeast chorismate mutase is a kind of variation that has been observed in two other allosteric enzymes. In aspartate transcarbamylase, a hrther 0 1998 IUPAC, Pure and Applied Chemistry 70,527-531

Yeast chorismate mutase 53 1 expansion along the three-fold axis and hrther rotation of the catalytic trimers relative to one another has been observed in a low angle X-ray study in solution (ref 16). And, in hemoglobin the T to R equilibrium has been extended: T to R to R2. In the R2 structure there are two water molecules not previously seen in the a& interface, and the R2 structure is proposed as the filly ligated state (refs. 17-20). Acknowledgement The authors thank the Organizers of the Conference, and the National Institutes of Health for Grant GM06920. We also acknowledge our collaboration with the research group of Professor G. Braus, including G. Schnappauf and R. Graf, for supplying the pure enzyme and the mutants. REFERENCES 1. G. H. Braus, MicrobiologicalReviews 55,349-370 (1991). 2. N. StrSlter, G. Schnappauf, G. Braus and W. N. Lipscomb, Structure, submitted 1997. 3. W. N. Lipscomb, Chemtracts-Biochemistry andmolecular Biology 2, 1-15 (1991). 4. W. N. Lipscomb,Adv. Enzymol. 68,67-151 (1994). 5. Y. Zhang, J-Y Liang, S. Huang and W. N. Lipscomb, J. Mol. Biol. 244,609-624 (1994). 6. T. Schirmer and P. R. Evans, Nature 343, 140-145 (1990). 7. P. R. Evans, Currrent Opinion in Structural Biology 1,773-779 (1991). 8. L. N. Johnson andd. Barford Jr.,J. Biol. Chem. 265,2409-2412 (1990). 9. M. F. Perutz, Proc. Roy. SOC. London B208, 135-162 ((1980). 10. J. Baldwin and C. Chothia, J. Mol. Biol. 129, 175-220 (1979). 11. M. F. Perutz, Mechanism of Cooperativity and Allosteric Regulation in Proteins, Cambridge University Press, Cambridge, England. 12. S. Iwata, K. Kamaq S. Yoshida, T. Minowa and T. Ohta, Nature Structural Biol. 1, 176-185 (1994). 13. N. Strtiter, K. HSkansson, G. Schnappauf, G. Braus and W. N. Lipscomb, Proc. Natl. had. Sci. USA 93, 3330-3334 (1996). 14. T. Schmidheini, H.-U. Mtisch, J. N. S. Evans and R. Braus, Biochemistry 29,3660-3668 (1990). 15. A. G. Grant, D. J. Schuller and L. J. Banazak, Protein Science 5,34-41(1996). 16. D. I. Svergq C. Barkerato, M. H. J. Koch, L. Fetter and P. Vachette, Proteins: Structure, Function and Genetics 27, 110-117 (1997). 17. F. R. Smith, E. E. Lattmanand C.W. Carter, Proteins 10, 81-91 (1991). 18. M. M. Silva, P. H. Rogers and A. hone, J. Biol. Chem. 267, 17248-17256 (1992). 19. F. R. Smith and K. C. Simmons, Proteins, 18,295-300 (1994). 20. M. A. Schumacher, E. E. Zhelemova, K. S. Poundstone, R. Kluger, R. T. Jones and R. G. Brennan, Proc. Natl. Acad Sci. USA 94,7841-7844 (1997). 0 1998 IUPAC, Pure and Applied Chemistry 70, 527-531